PROJECT SUMMARY Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide and is tightly linked to numerous metabolic diseases. Lipid droplet (LDs) are the major storage organelles of intracellular triacylglycerol (TAG) and, therefore, are the defining characteristic of NAFLD. In contrast to historical views of LDs as simply inert forms of energy storage, recent studies have identified LDs as important organelles that influences cellular function and signaling in addition to the regulation of TAG content and turnover. Given the prevalence of NAFLD and its massive burden worldwide, understanding the mechanisms governing LD metabolism is paramount if we aim to progress to effective dietary, lifestyle or pharmaceutical therapies targeting NAFLD and its comorbidities. This application will build upon recent findings from our laboratory regarding the role of adipose triglyceride lipase (ATGL) in liver energy metabolism. Our published and preliminary data show that ATGL is a major hepatic lipase that promotes oxidation of hydrolyzed fatty acids (FAs) and links cAMP/PKA signaling to sirtuin 1 (SIRT1)-mediated induction of PGC-1a and its transcriptional binding partners (e.g. PPAR-a and FoxO1). Once activated in response to ATGL, SIRT1 promotes autophagy/lipophagy, which in turn is responsible for bulk degradation of hepatic LDs and the subsequent efflux of FAs. Based on this data, the objective of this proposal is to elucidate the physiological and mechanistic regulation of lipophagy and trafficking of lysosome-derived FAs. We hypothesize that ATGL, via intracellular trafficking of oleate, activates SIRT1 to control specific arms of lipophagy leading to LD catabolism and lysosomal-mediated FA efflux. To test our hypothesis, we will conduct the following specific aims: 1) to characterize the physiological regulation of and interplay between ATGL and lipophagy/FA efflux; 2) to define the mechanism linking ATGL to SIRT1 signaling and lipophagy induction; and 3) to elucidate the regulation and consequences of lysosomal-mediated FA efflux on hepatic energy metabolism. Aim 1 will employ cell, perfused liver and mouse models to characterize how physiological factors regulate lipophagy. Aim 2 will use both cell and mouse models to dissect out the signaling network linking ATGL to the regulation of autophagy/lipophagy and FA efflux. Aim 3 will focus on lysosome fusion to the plasma membrane as a therapeutic target to increase lipophagy. These studies are innovative in that they will answer novel questions about the regulation of LD catabolism and signaling. This work is significant because it advances our understanding of the defining characteristic of NAFLD (i.e. LDs), which will have a positive and sustained impact towards the goal of preventing or treating NAFLD and related comorbidities.